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GABA, The Major Inhibitory Neurotransmitter in the Brain

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Hinton Tina, and Johnston Graham A.R. (2018) GABA, The Major Inhibitory Neurotransmitter in
the Brain. Reference Module in Biomedical Sciences. Elsevier. 5-Oct-18 doi: 10.1016/B978-0-12-
801238-3.96594-2.

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 Author's personal copy

GABA, The Major Inhibitory Neurotransmitter in the Brain

Tina Hinton and Graham AR Johnston, The University of Sydney, Sydney, NSW, Australia
© 2018 Elsevier Inc. All rights reserved.

Introduction 1
GABA Conformations 1
GABA Synthesis 1
GABA Release 2
GABA Receptors 3
GABAA Receptors 3
GABAA receptors and tonic inhibition 3
GABAA receptor antagonists 4
GABAA receptor agonists 5
Modulation of GABAA receptors 5
Benzodiazepine and related agents 5
Flavonoids 7
Steroids and stress 8
Ethanol 10
GABAB Receptors 10
GABAC Receptors (Also Known as GABAA-r or GABA-r Receptors) 11
GABA Transport 11
GABA Metabolism 12
GABA Beyond the Central Nervous System 12
Conclusion 13
Acknowledgment 13
References 13

Introduction

Simple molecule, complex function: GABA (derived from the old name g-aminobutyric acid for 4-aminobutanoic acid) is a
neurotransmitter of major significance in the brain, being released by up to 40% of neurons to activate chloride channels resulting
generally in inhibition of firing (Bowery and Smart, 2006). GABA was first identified in the brain in 1950 following its earlier
discovery in microbes and plants (Fig. 1).
GABA has multiple roles in the brain. GABA is present in large amounts in the brain, distributed in distinctly different cellular
pools reflecting its multiple functions (Waagepetersen et al., 1999). Its ubiquitous presence in the brain and spinal cord means that
almost all neurons either release GABA, express GABA receptors, or are innervated by neurons that do. Thus, most brain functions
are likely to involve GABA as a neurotransmitter. In addition to its role as a neurotransmitter of major significance, it functions as an
important metabolite and a neurotrophic agent.
GABA mechanisms are thought to be involved in anesthesia, anxiety, epilepsy, memory, neurodegenerative disorders, schizo-
phrenia, sleep, stress, cardiovascular and neuroendocrine function. Many agents act through GABA mechanisms including barbi-
turates, benzodiazepines, ethanol, flavonoids, general anesthetics, and neuroactive steroids (Froestl, 2011).

GABA Conformations

GABA interacts with a variety of binding sites on enzymes, receptors, and transporters. The conformational flexibility of GABA is
considered to be important for these interactions. Conformationally restricted analogs of GABA are able to selectively interact with
different GABA binding sites. Their activity indicates that different conformations of GABA interact with the different binding sites,
with the conformationally flexible GABA key able to uniquely interact with the different locks.
The conformational flexibility of GABA may be restricted by incorporation of unsaturated carbon–carbon bonds in the GABA
backbone, as in cis- and trans-4-aminocrotonic acid, or binding the backbone carbon–carbon bonds with a ring structure, as in
CAMP, or both as in muscimol and THIP (Fig. 2). Conformational restriction may also be achieved by addition of a heavy group
such as in baclofen. These agents each have selective actions on aspects of the GABA system (Johnston, 2014, 2016).

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2 GABA, The Major Inhibitory Neurotransmitter in the Brain

Fig. 1 GABA, zwitterionic structure and different low energy conformations (shapes).

Fig. 2 Conformationally restricted analogs of GABA and their selective actions on aspects of the GABA system.

GABA Synthesis

GABA is synthesized from L-glutamate via a-decarboxylation by glutamate decarboxylase (GAD). As the rate-limiting enzyme for
GABA synthesis, inhibition of GAD results in a decrease in GABA levels in the brain resulting in an increase in neuronal excitation
and ultimately convulsions.
Glutamine in astrocytes is a major source of GABA via glutamate, as inhibition of glutamine transport reduces GABA levels.
Other sources of GABA may include homocarnosine, ornithine, and putrescine.
GAD exists in two isoforms with different molecular weights, subcellular distributions, and regulatory properties encoded by
different genes on different chromosomes (Waagepetersen et al., 1999). GAD65 is associated primarily with vesicles in GABA-
containing nerve terminals, therefore it is thought to be involved in the synthesis of GABA mediating fast synaptic inhibition. GAD67
is more widely distributed in cytoplasm, found in neuron terminals and predominantly cell bodies, and is involved in the metabolic
function of GABA via the tricarboxylic acid cycle (Waagepetersen et al., 1999).
Anti-GAD antibodies are associated with autoimmune neurological disorders as well as type 1 diabetes as a result of GABA being
expressed in b-cells of the pancreas (Mitoma et al., 2017). Disorders of GAD are found in schizophrenia and bipolar disorder.

GABA Release

The vesicular release of GABA from presynaptic terminals following depolarization is calcium-dependent and is blocked by tetanus
toxin, a convulsant that also blocks the release of glycine, an important inhibitory transmitter in the spinal cord. GABA is also
released from other than presynaptic terminals, for example, from astrocytes by reverse action of GABA transporters or by other
nonvesicular release mechanisms (Koch and Magnusson, 2009). GABA may act as a “gliotransmitter” released from glial cells to
activate GABA receptors on neighboring neurons (Yoon and Lee, 2014).

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GABA, The Major Inhibitory Neurotransmitter in the Brain 3

GABA Receptors

Synaptic receptors for GABA may be divided into two classes based on their mechanism of action. Ionotropic GABA receptors act as
ligand-gated chloride channels. Metabotropic GABA receptors act as G protein-coupled receptors. These two classes were originally
termed GABAA and GABAB receptors on the basis of sensitivity to the convulsant alkaloid, bicuculline, and the central nervous
system depressant and skeletal muscle relaxant, baclofen. GABAA receptors are antagonized by bicuculline and insensitive to
baclofen while GABAB receptors are insensitive to bicuculline and sensitive to baclofen. This division has however been confounded
by the finding of ionotropic receptors that are insensitive to both bicuculline and baclofen. These receptors have been widely called
GABAC receptors, based on their distinctive structure, pharmacology, and physiology.

GABAA Receptors
These receptors belong to the superfamily of CYS-loop receptors that include ionotropic receptors for acetylcholine (nicotinic
receptors), glycine and 5-hydroxytryptamine (5HT3 receptors). GABAA receptors are heteromeric complexes made up of five protein
subunits, usually of three different types of subunits. In general, each subunit has a large extracellular N-terminal domain containing
the GABA binding site, a short extracellular C-terminal and four transmembrane domains with the second transmembrane forming
the walls of a chloride channel in combination with the other protein subunits. Activation of the chloride channel in mature
neurons results in the influx of chloride ions which stabilizes the membrane potential, thereby reducing excitatory depolarization of
the postsynaptic membrane. Intracellular chloride concentrations are regulated by the sodium–potassium–chloride cotransporter
NKCC1 and potassium-chloride cotransporter 2 (KCC2). Developmental changes in the cotransporters can mean that GABA has a
predominately depolarizing action in immature neurons changing to a hyperpolarizing action at maturity. The shift in action can be
influenced by a variety of factors including neonatal maternal separation (Furukawa et al., 2017), amyloid precursor protein
(Doshina et al., 2017), and immune activation (Skilbeck et al., 2018).
A total of 16 GABAA subunits have been identified in humans: six a-subunits, three b-subunits, three g-subunits, and d-, e-, y-,
and p-subunits. In addition there are splice variants and point mutations. Three r-subunits have been described that are sometimes
classified as GABAA receptor subunits on the basis of sequence similarity but these are more frequently classified as GABAC receptor
subunits. Different combinations of subunits result in different properties of GABAA receptors (Möhler, 2015). Thus there are a
myriad of subunit combinations making up a huge variety of possible GABAA receptors, making them the most complex of
neurotransmitter receptors. The most abundant pentameric isoform contains two a1-, two b2-, and one g2-subunits. Studies using
concatenated subunits indicate that the subunits are arranged g2–b2–a1–b2–a1 counterclockwise forming the ion channel as
viewed from the cell exterior (Sigel and Steinmann, 2012). Recombinant receptor combinations expressed in vectors such as Xenopus
oocytes together with in vivo gene knock out and knock in studies show that generalizations may be made concerning particular
combinations. Thus, activation of a1bg2 GABAA receptors mediates sedation, amnesia and anticonvulsant effects, whereas a2bg2
receptors mediate anxiolytic and myorelaxant effects and a5bg2 receptors influence learning and memory.
The mix of GABAA receptor subunits can be altered as in the so-called alpha subunit switch whereby a1- and a2-subunits exhibit
age-dependent expression. In general, a1-subunits predominate in adult brain and a2-subunits are more abundant in younger
tissue (Davis et al., 2000). Early-life environment has long-lasting effects on the mix of GABAA receptor subunits influencing adult
behavior (Skilbeck et al., 2018).
Subtype selective agents are being intensively sought in order to overcome the relative lack of selectivity of many therapeutic
agents acting on GABAA receptors. For example, unlike benzodiazepines, an anxiolytic agent devoid of addictive, amnesia and
sedative effects would be highly desirable (Skolnick, 2012). A very successful new therapeutic agent is S44819, a highly selective
competitive antagonist of GABAA receptors that contain the a5-subunit (Etherington et al., 2017). It is an orally active cognitive
enhancer acting at the orthosteric site at the a–b interface on extrasynaptic GABAA receptors to reduce tonic inhibition. Other agents
are known to act as negative allosteric modulators by binding to the benzodiazepine recognition site between at the a5–g2 subunits.
Agents that are selective for a5 subunit-containing GABAA receptors enhance cognitive performance in a variety of animal models
without sedative or proconvulsive effects.

GABAA receptors and tonic inhibition

Extrasynaptic GABAA receptors mediate a long lasting tonic inhibition that is not under direct synaptic control (Belelli et al., 2009).
Tonic GABAA receptors can be activated by GABA released from glial cells or dendrites and from synaptically released GABA not
cleared efficiently from the extracellular space. Tonic inhibition might be considered as regulating neuronal excitability, network
oscillations, neuronal development, and cognition. It may be responsible for generation of a major percentage of the total
inhibitory charge on certain neurons. GABAA receptors represent a target for antiepileptic therapy and cognitive enhancement
(Mortensen et al., 2010).
Subtypes of GABAA receptors mediating tonic inhibition preferentially contain a4-, a5-, a6-, and d-subunits whereas g2 subunits
are found preferentially in synaptic receptors. In general, tonic GABAA receptors are activated at lower concentrations of GABA and
desensitize more slowly than synaptic receptors.
Agents that selectively target tonic d-containing GABAA receptors include THIP (Gaboxadol) that influences sleep, and the
anesthetics alfaxalone and propofol.

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4 GABA, The Major Inhibitory Neurotransmitter in the Brain

GABAA receptor antagonists

The most widely used GABAA receptor antagonists as experimental tools are bicuculline, gabazine, and picrotoxinin (Fig. 3). Each
produces convulsions on systemic administration but they act in significantly different ways. Bicuculline is the defining antagonist
for GABAA receptors (Johnston, 2013). Bicuculline and gabazine are competitive antagonists interacting with different residues on
GABAA receptors. Picrotoxinin is a noncompetitive antagonist acting on the chloride channel of GABAA receptors and on several
other ionotropic CYS-loop receptors including glycine, GABAC and 5HT3 receptors.
Many structurally diverse agents are known to act as GABAA receptor antagonists providing opportunities for the discovery of
subtype selective antagonists. Nonconvulsive antagonists are of therapeutic interest for the treatment of cognitive problems, myopia
and other CNS disorders.
Picrotoxin was the first reported GABAA receptor antagonist. It is a 50:50 mixture of picrotoxinin and picrotin. Picrotoxinin
(Fig. 3) is a convulsant as are several other terpenoids including tutin. Some structurally related terpenoids from Ginkgo biloba, such
as bilobalide, that, although acting as GABAA receptor antagonists, are not convulsants (Ng et al., 2017). These terpenoids have
therapeutic use in the treatment of memory impairment (Manayi et al., 2016).
Bicuculline (Fig. 3) is selective for GABAA receptors, having little effect on GABAB, GABAC, glycine, and 5-HT3 receptors
(Johnston, 2013). Its action is largely independent of GABAA subunit composition, binding at the orthosteric site to stabilize the
receptors in a closed state. Single channel studies show that by competing with GABA for its binding site, bicuculline acts to reduce
both chloride channel open time and opening frequency. Bicuculline is slowly converted to bicucine, a much less active convulsant
in solution at neutral pH (Olsen et al., 1975). Acidic pH slowly reverses this transformation and thus bicuculline solutions should
always be freshly prepared in order to preserve maximum convulsant potency. Quaternary salts of bicuculline, such as bicuculline
methiodide (“N-methyl bicuculline”) or methochloride, are much more stable than bicuculline, but they do not cross the blood
brain barrier on systemic administration. The quaternary salts differ in their pharmacology to bicuculline itself in that they are much
less selective. It is not always clear in many publications whether the investigators use bicuculline or a quaternary salt. The
quaternary salts have significant actions on nicotinic receptors, calcium-activated potassium channels and acetylcholinesterase.
Thus, while ensuring chemical stability of bicuculline, the quaternary salts may be less effective tools owing to their reduced binding

Fig. 3 Antagonists that distinguish between the three major classes of GABA receptors.

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GABA, The Major Inhibitory Neurotransmitter in the Brain 5

specificity for GABAA receptors. Subject to these considerations, bicuculline and its quaternary salts continue to be used extensively
as GABAA receptor antagonists in experimentation.
Gabazine (Fig. 3, also known as SR 95531) is a relatively specific, potent and competitive antagonist of GABAA receptors
(Heaulme et al., 1986). Structural analogs of gabazine have been developed as more potent agents (Iqbal et al., 2011). Gabazine
analogs incorporating photoactive groups, such as GZ-B1, have been developed as photo-activated antagonists of GABAA receptors
(Mortensen et al., 2014). These antagonists provide dynamic tools for visualizing GABAA receptors, permitting a novel means of
investigating receptor location, function, and trafficking (Mortensen et al., 2014).
S44819 (Fig. 3, Egis-13,529), is a novel oxazolo-2,3-benzodiazepine derivative that selectively inhibits GABAA receptors that
contain the a5-subunit (Etherington et al., 2017). It appears to act as a competitive antagonist at the orthosteric site at the a–b
subunit interface of GABAA receptors containing only a5 subunits and enhances cognitive performance in a variety of animal
models without sedative or proconvulsive effects. S44819 has been shown in healthy young humans to be orally active, reaching the
cerebral cortex on oral administration where it increases cortical excitability, acting on extrasynaptic receptors to reduce tonic
inhibition (Darmani et al., 2016). S44819 and related a5 selective agents show promise as therapeutic agents.
Other important classes of antagonists include agents derived from 4-PIOL (Krall et al., 2015). Of particular interest is DPP-4-
PIOL that selectively antagonizes tonic over phasic GABAergic currents in the hippocampus, suggesting a degree of subunit
specificity (Boddum et al., 2014). Salicylidene salicylhydrazide has been reported as a potent antagonist of GABAA receptors
containing the b1 subunit using a high throughput screen (Thompson et al., 2004). The most potent GABAA receptor antagonist is
the convulsant steroid derivative RU5135, being some 500 times more potent than bicuculline (Hunt and Clementsjewery, 1981). It
acts as a competitive antagonist, sharing a common site of action with bicuculline. However, it lacks specificity, as it is also a glycine
receptor antagonist sharing a common site of action with strychnine (Simmonds and Turner, 1985). Terpenoids isolated from Salvia
triloba including ursolic acid, carnosol, oleanolic acid, and rosmanol act as nonconvulsant GABAA receptor antagonists (Abselhalim
et al., 2014).

GABAA receptor agonists

Muscimol (Fig. 2), a psychoactive isoxazole from Amanita muscaria mushrooms, is widely used as a selective GABAA agonist
(Johnston, 2014). It is inactive at GABAB receptors and is a more potent partial agonist at GABAC receptors. It is a weak substrate for
GABA uptake and is inactive in relation to GABA transaminase. Its effect on GABAC receptors means that muscimol actions should
not be interpreted solely in relation to GABAA receptors unless shown to be completely blocked by bicuculline and insensitive to
TPMPA. Muscimol was the lead compound in the discovery of important GABAergic agents including nipecotic acid, tiagabine,
THIP and 4-PIOL by medicinal chemists in Denmark (Krall et al., 2015).
THIP (Gaboxadol) is a bicyclic analog of muscimol (Fig. 2). It acts as a GABAA agonist on most GABAA receptor subtypes, except
at extrasynaptic receptors where it acts, like muscimol, as a super agonist (Mortensen et al., 2010). These authors state “The ability to
specifically increase the efficacy of receptor activation, by selected exogenous agonists over that obtained with the natural
transmitter, may prove to be of therapeutic benefit under circumstances when synaptic inhibition is compromised or
dysfunctional.” THIP acts as a GABAC receptor antagonist (Naffaa et al., 2017) and has shown activity as an analgesic as well as a
novel type of hypnotic that increases non-REM sleep and enhances delta activity (Krogsgaard-Larsen et al., 2004).
Endogenous agonists at GABAA receptors include taurine, GABOB (b-hydroxyGABA), b-alanine, and methylglyoxal. Methyl-
glyoxal is a partial agonist at GABAA receptors. This byproduct of glycolysis is anxiolytic in mice (Distler et al., 2012). Inhibitors of
the enzyme glyoxalase 1, that metabolizes methylglyoxal, show promise as faster acting antidepressants by reducing the amount of
methylglyoxal acting of GABAA receptors (McMurray et al., 2018).
A selective agent for the activation of GABAA receptors without effect on GABAB or GABAC receptors is 4-aminotetrolic acid
(Fig. 2), an analog of GABA containing a triple bond that holds it in a fully extended conformation (Johnston, 2016). It is an
inhibitor of GABA uptake and of GABA transaminase.

Modulation of GABAA receptors

There are a multitude of agents that act as allosteric modulators of GABAA receptor function. Such modulators are much sought after
for their potential subtype selectivity as a result of the greater structural diversity of allosteric sites as distinct from the orthosteric
GABA binding sites (Changeux, 2013; Christopoulos, 2002). These agents include benzodiazepines, flavonoids, and steroids
(Möhler, 2015; Hanrahan et al., 2015). Many anesthetic agents, including volatile anesthetics, barbiturates, etomidate, propofol,
long-chain alcohols, and neurosteroids act in the transmembrane domain of GABAA receptors as positive allosteric modulators
(Olsen, 2015). Multiple binding sites are involved that may overlap or may be allosterically linked. Of particular interest are
extensive studies on etomidate and propofol (Maldifassi et al., 2016).
New concepts about receptor modulation are recognized. Terms such as positive (PAM), negative (NAM), and silent (SAM)
allosteric modulator were used to describe different types of modulator. Silent allosteric modulators are also called neutralizing
allosteric modulators. There are also allosteric agonists, and second order modulators have been described that modulate the action
of first order modulators such as benzodiazepines.

Benzodiazepine and related agents

In the 1970s benzodiazepines, such as diazepam (Valium), were the most widely prescribed drugs. The discovery that their therapeutic
effects were related to modulation of GABA receptors profoundly influenced the study of these receptors (Möhler, 2015). A large

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6 GABA, The Major Inhibitory Neurotransmitter in the Brain

range of structurally diverse chemicals was found to interact with benzodiazepine modulatory sites on GABAA receptors, leaving
GABAB and GABAC receptors largely unaffected.
Benzodiazepines have no direct action on GABAA receptors. Instead, they enhance the action of GABA by increasing the
frequency of ion channel openings. In contrast, barbiturates and ethanol increase the duration of channel openings and thus
increase the maximal effect of GABA. Barbiturates also have an allosteric agonist action on GABAA receptors, in other words they can
directly activate the receptor at high concentrations. As a result they are much more dangerous drugs than benzodiazepines, thus
benzodiazepines have replaced barbiturates as therapeutic agents. Agents such as DMCM have the opposite effect, decreasing the
action of GABA. This incorrectly led to the concept of “inverse agonist.” DMCM is correctly described as a negative allosteric
modular. True inverse agonists act at constitutively active G protein-coupled receptors (Fig. 4).
Benzodiazepines bind at the interface of the a- and g-subunits. The a-subunit requires a histidine residue as in a1-, a2-, a3-, and
a5-subunits. GABAA receptors containing a1-subunits are associated with stronger hypnotic and sedative effects of benzodiazepines,
while those containing a2- and a3-subunit are stronger anxiolytics (Crestani and Rudolph, 2015; Fritschy, 2015). Receptors
containing a5-subunits are associated with memory (Etherington et al., 2017). The therapeutic potential for agents acting selectively
on GABAA receptor subtypes is considerable to areas such as affective disorders, autism, Down syndrome, and schizophrenia
(Rudolph and Möhler, 2014).
Caution should be used in interpreting the many studies using flumazenil as a probe for GABAA receptor activity as not all
GABAA receptors are sensitive to this silent allosteric modulator. Such studies generally only pick up GABAA receptors containing g2-
subunits. Benzodiazepines have been shown to act on GABAA receptors by “two distinct and separable mechanisms” (Walters et al.,
2000). At low concentrations (nanomolar) they modulate in a manner sensitive to flumazenil, whereas at higher concentrations
(micromolar) they act in a flumazenil-insensitive manner. The high affinity action is confined to GABAA receptors containing the
g2-subunit. In adult brain, g2-subunits are much more abundant that g1 or g3. When g1 replaces g2, flumazenil changes from a
silent allosteric modulator to a positive allosteric modulator. Transgenic g2 knockout mice are insensitive to low doses of
benzodiazepines. The significance of the flumazenil-insensitive, low affinity site of benzodiazepine action remains somewhat
enigmatic (Walters et al., 2000). It may be related to the flumazenil-insensitive actions of many flavonoids (Hinton et al., 2017), as
will be discussed below.
Initially described as a peripheral benzodiazepine receptor, translocator protein (TSPO) binds a variety of benzodiazepine-like
drugs and is involved in cholesterol transport across the outer mitochondrial membrane in many tissues. It is a target for anxiety and
neurological disorders (Nothdurfter et al., 2012).
So-called nonbenzodiazepines that bear no obvious chemical resemblance to benzodiazepines have been developed for
insomnia, including drugs such as zolpidem. These “Z-drugs” act as positive allosteric modulators of a1-containing GABAA
receptors, binding to the benzodiazepine binding site. Their relative selectivity offers some advantages over benzodiazepines, but
they are not without their problems (Gao and Smith, 2010).
The term endozepine is used to describe endogenous compounds with effects similar to benzodiazepines (Farzampour et al.,
2015). They include the protein DBI (diazepam binding inhibitor) and oleamides. Endogenous cannabinoids also positively
modulate GABAA receptors (Sigel et al., 2011; Bakas et al., 2017).

Fig. 4 Agents related to benzodiazepines that modulate GABAA receptors.

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GABA, The Major Inhibitory Neurotransmitter in the Brain 7

Flavonoids
In the 1990s, scientists in Argentina described flavonoids as “a new family of benzodiazepine receptor ligands” (Medina et al.,
1997). They have proved to be much more than that, far surpassing the benzodiazepines in their range of actions of GABAA
receptors (Hanrahan et al., 2015). Flavonoids are consumed extensively in our diet. Many are able to cross the blood brain barrier
into the brain and constitute agents capable of influencing the function of GABAA receptors. In addition to the large range of
flavonoids that occur naturally, there are a considerable number of synthetic flavonoids that influence GABAA receptors. Natural
and synthetic flavonoids are known to act as positive, silent and negative allosteric modulators as well as allosteric agonists (Hinton
et al., 2017). Many of these actions are insensitive to flumazenil.
The flavones hispidulin, luteolin, and apigenin (Fig. 5) are closely related structurally but show differing profiles of activity at
GABAA receptors and differing effects in vivo, demonstrating that small differences in chemical structure have profound effects on
the biological properties of these agents.
Hispidulin is an anticonvulsant from sage (Salvia officinalis) that acts as a positive allosteric modulator on recombinant a1b2g2
GABAA receptors that is only partially blocked by flumazenil (Kavvadias et al., 2004). Like many flavonoids, it exhibits a biphasic
action and is approximately equipotent at each of six different a-subunit containing GABAA receptors—a1,2,3,5,6b2g2S, enhancing
at low concentrations (EC50 0.8–5 mM) and inhibiting at higher concentrations (>30 mM).
Luteolin, found in many plants including green peppers and celery, has an anxiolytic and sedative action on experimental
animals that is insensitive to flumazenil, consistent with acting as a positive allosteric modulator at GABAA receptors (Hinton et al.,
2017). Although structurally related to hispidulin, it does not act as an anticonvulsant. Its association with GABAA receptors is
suggested by the ability to promote GABA-mediated chloride influx in human neuroblastoma cells, which is attenuated by the
GABAA receptor antagonist bicuculline.
Apigenin, an anxiolytic found in chamomile, has complex modulatory actions on GABAA receptors. Most interesting is the
ability of apigenin to modulate the first order modulation of GABAA receptors by benzodiazepines such as diazepam (Campbell
et al., 2004). This second order modulation is selective for diazepam first order modulation—it has not been observed for
pentobarbitone or allopregnanolone modulation. Second order modulation has been noted in other systems (Mesce, 2002; Ribeiro
and Sebastiao, 2010) and may represent an obscure novel mechanism of drug action deserving further investigation, with the
potential to lead to decreased therapeutic doses.

Fig. 5 Some naturally occurring flavonoids that influence GABAA receptors.

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8 GABA, The Major Inhibitory Neurotransmitter in the Brain

(
)-Epigallocatechin gallate (EGCG, Fig. 5), the major flavonoid in green tea (Camellia sinensis), is an order of magnitude more
potent than apigenin as a second order modulator (Campbell et al., 2004). In addition, it was been found to reverse b-carboline
negative allosteric modulation of GABA action (Vignes et al., 2006). This indicates that EGCG may act as a second-order modulator
with respect to the first-order modulation by both positive and negative allosteric modulators that act via benzodiazepine-binding
sites on GABAA receptors. It has effects on learning and memory, and demonstrates dose-dependent stress-reducing, anxiolytic,
sedative and hypnotic properties in a number of animal models.
A series of flavan-3-ol esters were synthesized as simpler analogs of EGCG (Fernandez et al., 2008). Fa131 (Fig. 6) proved to be a
nonsedating anxiolytic and a selective positive allosteric modulator of a2-containing GABAA receptors. Fa131 is the first positive
allosteric modulator to distinguish between a2- and a3-containing GABAA receptors, highlighting the potential of targeting
flumazenil-insensitive allosteric sites in the search for new anxio-selective drugs.
The structurally related Fa173 acts as a silent allosteric modulator, blocking the actions of Fa131 and of etomidate, loreclezole,
and low affinity diazepam, without influence on propofol, thiopental, and allopregnanolone (Fernandez et al., 2012). This suggests
that Fa131, etomidate, loreclezole, and low (nonflumazenil-sensitive) doses of diazepam all exert their positive modulatory effects
via a common or overlapping binding site that can be blocked by the silent allosteric modulator Fa173.
Among the many synthetic flavonoids that influence GABAA receptors are a series of 6-substituted flavones (Hanrahan et al.,
2015). These illustrate that highly structurally similar flavones can act as flumazenil-sensitive and flumazenil-insensitive positive
allosteric modulators, silent allosteric modulators, and negative allosteric modulators.
These and other studies have identified the presence of multiple sites on GABAA receptors at which a variety of flavonoids can act
as modulators. The sites include ones that are insensitive to the classical benzodiazepine silent allosteric modulator flumazenil and
are described as low affinity benzodiazepine sites. Perhaps, these would be more appropriately described as flavonoid sites as they
appear to be activated by numerous natural and synthetic flavonoids (Hinton et al., 2017).

Steroids and stress

A variety of steroids modulate GABAA receptors (Lambert et al., 2009). These include neurosteroids, that is, steroids that are made in
the brain, and neuroactive steroids, such as corticosteroids and glucocorticoids. Many of these steroids are involved in the response

Fig. 6 Some synthetic flavonoids that influence GABAA receptors.

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GABA, The Major Inhibitory Neurotransmitter in the Brain 9

to stress. Sex differences observed in the GABAergic neurotransmitter system following acute stress indicate the involvement of
gonadal steroids (Skilbeck et al., 2010).
The essential role for GABA in stress is well established, and the GABAergic system represents an important modifiable system for
regulating stress and anxiety. In addition to central nervous system regulation, GABA modulates stress via the fast acting sympatho-
adrenal-medullary (SAM) system, as part of the autonomic nervous system, and the slower acting hypothalamic–pituitary–adrenal
(HPA) axis.
Endogenous neurosteroids (Fig. 7) such as 3a-hydroxy-5a-pregnan-20-one (allopregnanolone, or 3a,5a-THP), synthesized from
progesterone, and 3a,21-dihydroxy-5a-pregnan-20-one (allotetrahydrodeoxycorticosterone, or 3a,5a-THDOC), synthesized from
corticosterone, are also known to be potent positive modulators of GABAA receptors (Hosie et al., 2009). These neurosteroids
modulate many GABAA receptor isoforms including combinations of ab, abg, and abd, with apparently greater potency at abd
receptor types (Lambert et al., 2009). It is likely that multiple binding sites for steroids exist on the GABAA receptor complex. These
neurosteroids can also be synthesized from cholesterol de novo in the brain during stress (Barbaccia et al., 2001; Higashi et al.,
2005). Synthetic steroids, including anesthetics such as alfaxalone and ganaxolone (Belelli and Lambert, 2005), also act as allosteric
modulators of the GABAA receptor.
Glucocorticoids, in addition to their genomic mechanism of action via intracellular glucocorticoid receptors, also have
nongenomic effects through modulation of GABAA receptors (Purdy et al., 1991). Cortisol and cortisone act as bi-directional
modulators of agonist binding and GABA function at GABAA receptors, with enhancement of agonist binding and GABA-mediated
response at low concentrations (pM–nM) and inhibition at higher concentrations (nM–mM) (Ong et al., 1987).
Acute stress in rodents has been shown to induce rapid changes in the GABAergic system. The number of functional binding sites
for GABA following a 3 min swim stress in mice was altered in opposite directions in males and females. These large changes were
rapid in onset but not sustained. This suggests a mechanism for rapid GABAergic plasticity possibly via receptor trafficking or
changes in endogenous GABAergic substances (Skilbeck et al., 2010).
Under chronic stress conditions it appears that neurosteroid concentrations decrease (Serra et al., 2000), while GABAA receptor
expression and function is also altered (Skilbeck et al., 2010, 2018), possibly contributing to disinhibition of the HPA axis and well-
documented elevated glucocorticoid concentrations following chronic stress.
Neurosteroids have been shown to enhance tonic GABAergic inhibition mediated by receptors containing the d subunit (Belelli
and Lambert, 2005). Chronic stress in adulthood decreases d subunit mRNA in the paraventricular nucleus of the hypothalamus
(Verkuyl et al., 2004) and, coupled with acute corticosteroid exposure, is found to increase a1 subunit mRNA in dentate gyrus
granule cells (Qin et al., 2004). In a recent investigation of early life conditioning impacts on GABAA receptor subunit expression, it
was shown that early life environment affects the regionally-dependent developmental subunit switch from a2 to a1 dominance,

Fig. 7 Steroid positive allosteric modulators of GABAA receptors.

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10 GABA, The Major Inhibitory Neurotransmitter in the Brain

and early life stress exaggerates this switch such that an increase in a1:a2 ratio occurred in stressed animals compared to the normal
condition in regions of the brain known to be involved in stress response such as the hippocampal dentate gyrus (Skilbeck et al.,
2018). This region thus represents a point of convergence for the impact of chronic stress on GABAergic regulation, its role in
neuropsychiatric disease development, and the potential role for neurosteroids in this response.
Given the important role of steroids in stress, and the essential role of stress in manifestation of a number of neuropsychiatric
disorders, including depression, anxiety and schizophrenia, chronic deregulation of steroid modulation of GABAA receptors is
thought to contribute to pathologies underlying such disorders (Cullinan et al., 2008). Some neurosteroids have been investigated
for their roles and potential therapeutic benefits in neuropsychiatric illnesses where stress is known to be a contributor (Schule et al.,
2011; Shirayama et al., 2011).

Ethanol
The key ingredient of regularly consumed “alcohol” is ethanol. Behavioral effects of ethanol include myorelaxation, anxiolysis,
sedation, impaired cognitive function, and an anticonvulsant effect. At higher doses, it can cause motor incoordination, ataxia,
amnesia, hypnosis, and anesthesia, and eventually respiratory depression and coma. While ethanol is known to influence several
sites in the central nervous system, it is now well understood that its major action at low to moderate doses is to positively modulate
GABAA receptors (Howard et al., 2014). At higher doses, ethanol also influences glutamate, glycine, and acetylcholine receptors.
Moreover, GABAA receptors are implicated in alcohol dependence, with studies showing polymorphisms in a2 and b1 subunits
confer vulnerability to alcohol dependence (McCabe et al., 2017).
At low to moderate doses (about 1–20 mM blood ethanol, 0.004%–0.08%, up to 2 standard drinks in 1 h), ethanol acts on
GABAA receptors containing the d subunit, coupled with the a4 and a6 subunits. The extra-synaptically-located a4bd and a6bd
receptors have greater affinity for GABA than the synaptic GABAA receptors, and, as described earlier, have been shown to regulate
the tonic inhibition mediated by ambient extracellular GABA. Ethanol administration increases neurosteroid concentration in the
periphery and CNS, with effects dependent on dose and region examined (Weiner and Valenzuela, 2006). Ethanol administration is
also shown to promote local neurosteroidogenesis (Sanna et al., 2004). Thus, ethanol influences GABAA receptor function in a
variety of ways—it positively modulates certain GABAA receptors in various brain regions, it promotes neurosteroid synthesis, and
neurosteroids form an indirect mechanism for ethanol action at GABAA receptors, by potentiating the effects of ethanol. Induction
of neurosteroid synthesis may augment the effects of ethanol since neurosteroids bind to d-containing GABAA receptor subunits and
produce anxiolytic, sedative, and anesthetic effects themselves.
At doses that begin to cause sedation and motor incoordination (about 20–30 mM), ethanol acts on GABAA receptors contain-
ing the g2L subunit (Howard et al., 2014). Receptors containing these subunits, when coupled with an a1, a2, a3, or a5 subunit, are
important for some of the sedative, anxiolytic, myorelaxant, anticonvulsant, and amnestic effects of GABA activation. At anesthetic
doses (50–400 mM) ethanol acts on most GABAA receptors (Howard et al., 2014). Ethanol also appears to enhance presynaptic
GABA release in the amygdala, hippocampus, and cerebellum, which may also contribute to its effects (Weiner and
Valenzuela, 2006).
The well-characterized flux in neurosteroid synthesis and expression of d subunit-containing GABAA receptors under conditions
of stress, during the menstrual cycle, and in neuropsychiatric disorders may explain the sex differences observed in the effects of
ethanol between males and females, as well as the differing effects of ethanol observed at different phases in the menstrual cycle
(Jimenez et al., 2017).

GABAB Receptors
The concept of GABAB receptors arose out of studies by David Hill and Norman Bowery in 1981 on the binding of the GABAB
analog baclofen (Fig. 2) to rat brain membranes (Hill and Bowery, 1981). They described a receptor that “differs from the classical
GABA site as it is unaffected by recognized GABA antagonists such as bicuculline.” They went on to state “We propose to designate
the classical site as the GABAA and the novel site as the GABAB receptor.” Special issues of Advances in Pharmacology and
Neuropharmacology that contain many authoritative papers on GABAB receptors have been published in honor of Norman Bowery
(Enna and Blackburn, 2010; Crunelli et al., 2018).
GABAB receptors are G protein-coupled receptors, formed as heterodimers composed of GABAB1 and GABAB2 protein subunits
(Enna and Blackburn, 2010; Froestl, 2011). They are tetramers with two GABAB1 subunits in close proximity flanked on either side
by GABAB2 subunits. Two isoforms of GABAB1 are known. The heterodimers formed by GABAB1a/GABAB2 proteins mediate
presynaptic inhibition whereas those formed by GABAB1b/GABAB2 mediate postsynaptic inhibition. GABA, GABAB agonists and
antagonists bind to the Venus flytrap module in the N-terminal region of the GABAB1 protein, while the GABAB2 mediates G protein
coupling. They are members of the group of class C G protein-coupled receptors with structural similarity to the metabotropic
glutamate receptors. Some GABAB receptors are coupled to activation of certain Kþ channels, producing slow inhibitory synaptic
currents. Other GABAB receptors can decrease Ca2þ conductance, regulate inositol triphosphate production, and/or inhibit cyclic
AMP production. GABAB antagonists, such as phaclofen (Fig. 3), have been described as well as positive and negative allosteric
modulators (Froestl, 2011).
CGP36742 (Fig. 3, also known as SGS742) was first described as an orally active GABAB antagonist for the treatment of cognitive
impairment (Froestl et al., 2004). Positive allosteric modulators show promise to treat addiction (Filip et al., 2015). GABAB agonists
may influence a range of disorders including trigeminal neuralgia, cerebral palsy, and autism (Froestl, 2011).

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GABA, The Major Inhibitory Neurotransmitter in the Brain 11

GABAC Receptors (Also Known as GABAA-r or GABA-r Receptors)

The concept of GABAC receptors arose out of studies of CACA (Fig. 2), a conformationally restricted analog of GABA in a folded
conformation. Initial studies in the spinal cord showed that CACA inhibited neuronal firing in a manner not consistent with the
action of either GABAA or GABAB receptors. Studies of the lack of effect of CACA on the binding of radioactive baclofen led to the
proposal that “folded analogs of GABA may interact with a class of binding site (GABAC?) insensitive to (
)-baclofen and
bicuculline” (Drew et al., 1984). Several groups described the action of CACA on bicuculline-insensitive, baclofen-insensitive
GABA receptors in the retina and receptors cloned from the retina. These became known as GABAC receptors made up of r-subunits
although the nomenclature of these ionotropic GABA receptors remains somewhat controversial (Naffaa et al., 2017). The
terminology GABAA-r is preferred by the IUPHAR Nomenclature Committee but is rarely used. GABA-r is being increasingly
used but the original GABAC terminology is by far the most popular, especially by workers who actually experiment on such
receptors, for example, in the retina. GABAC receptors are the least studied of the three classes on GABA receptors.
Functional GABAC receptors are found in the retina, spinal cord, hippocampus, superior colliculus, pituitary, and gastrointes-
tinal tract. Given the relatively restricted distribution of GABAC receptors in the CNS compared to GABAA receptors, GABAC
receptors may be a more selective drug target than GABAA receptors. The major indications for drugs acting on GABAC receptors
are in the treatment of visual, sleep, and cognitive disorders (Johnston et al., 2003; Naffaa et al., 2017).
GABAC receptors are members of the CYS-loop pentameric superfamily of ionotropic receptors. GABAC receptors are made up of
r1, r2, or r3 subunits. These subunits have considerable sequence similarity to GABAA, subunits, and also to glycine and nicotinic
subunits. GABAC receptors differ from GABAA receptors (a) in structure forming homomeric rather than heteromeric receptors,
(b) in physiology being 10–100 times more sensitive to GABA, slower in activation and deactivation, and less readily desensitized,
and (c) in pharmacology being readily differentiated by selective antagonists, having a unique agonist profile and being less readily
modulated by agents such as benzodiazepines (Naffaa et al., 2017). In addition, activation of GABAC receptors can have the
opposite effect to activation of GABAA receptors.
Whereas CACA is the classic selective agonist for GABAC receptors, it is only a partial agonist, (þ)-CAMP (Fig. 2), another analog
of GABA in a folded conformation, is a full agonist. TPMPA (Fig. 3) is a selective competitive antagonist for GABAC receptors that
does not influence GABAA or GABAB receptors (Murata et al., 1996). The GABAA antagonist bicuculline and the GABAB antagonist
phaclofen do not influence GABAC receptors. The channel blocker picrotoxinin inhibits both GABAC and GABAA receptors
indicative of similarities in the anion channels of both receptor types. The GABAA agonist THIP act as a GABAC antagonist. Aza-
THIP (Fig. 3) is a GABAC antagonist with negligible activity at GABAA receptors. These actions indicate that there are major
differences in the orthosteric binding sites on GABAC and GABAA receptors (Naffaa et al., 2017).
TPMPA and related GABAC antagonists have been patented for the treatment of myopia and memory disorders (Ng et al., 2011).
They have been used to study the role of GABAC receptors in various aspects of memory, sleep–waking behavior, and nociception.
The discovery that the GABAB antagonist CGP36742 was also a GABAC antagonist led to the development of cyclopentane analogs
that lacked GABAB activity but retained the GABAC activity. They enhanced memory and learning and inhibited the development of
myopia (Chebib et al., 2009). Selective GABAC antagonists may be therapeutic agents for the treatment of myopia, memory, and
sleep disorders.

GABA Transport

GABA is removed from the extrasynaptic environment by uptake via Naþ-dependent, high affinity GABA transporters into the
presynaptic terminal for re-release or into postsynaptic terminals and neighboring glial cells. Inhibitors of GABA transport may
prolong synaptic inhibition and act as anticonvulsants. The concentration gradients across membranes are large, with mM GABA
intracellularly and mM extracellularly. Several high affinity GABA transport proteins have been cloned (GAT1, GAT2, GAT3, and
BGT1), as well as a vesicular GABA transport protein (VGAT) representing important therapeutic targets (Schousboe et al., 2014;
Zhou and Danbolt, 2013).
GAT1 and GAT3 are the GABA transporters of major interest in the CNS. GAT1 is found predominately in GABAergic nerve
terminals where it recycles synaptically released GABA. GAT3 is found predominately in astrocytes where it maintains low
extracellular levels of GABA. GAT2 and BGT1 are expressed mainly in hepatocytes in the kidney and liver. The affinity of GABA
varies considerably with the reported Km values for the mouse isoforms being 8 mM for GAT1, 18 for GAT2, 0.8 for GAT3 and 80 for
BGT1. It is unlikely that BGT1 contributes to GABA inactivation.
The first therapeutically useful inhibitors of GABA transporters arose from studies on muscimol (Johnston, 2014; Krogsgaard-
Larsen et al., 2000). Structure–activity studies on analogs of muscimol led to the development of a series of nipecotic acid
derivatives of inhibitors of GABA transport, notably tiagabine (Fig. 8, Gabril), a GAT1 selective inhibitor that is anticonvulsant
and used in the treatment of epilepsy (Fig. 5). Tiagabine has profound side effects similar to those found in GAT1 knockout mice
(Schousboe et al., 2014; Zhou and Danbolt, 2013). It is the only GAT inhibitor approved for clinical use. Selective GAT3 inhibitors,
such as SNAP5114, are antinociceptive, acting by stimulation of GABAA and GABAB receptors in the spinal cord (Kataoka et al.,
2013). Novel highly selective GAT3 inhibitors have been developed (Damgaard et al., 2015) but there are as yet no reports of their
therapeutic properties. A GAT3 knockout mouse has been foreshadowed (Zhou and Danbolt, 2013) but no reports have emerged,
possibly as knocking out GAT3 may be lethal.

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12 GABA, The Major Inhibitory Neurotransmitter in the Brain

Fig. 8 GABA transport and enzyme inhibitors.

The vesicular transporter VGAT is important for the packing of synaptic vesicles with GABA, thereby determining the amount of
GABA released per vesicle (Van Liefferinge et al., 2013). No agents have been found that specifically inhibit VGAT although the
GABA aminotransferase inhibitor vigabatrin shows some activity. VGAT knockout mice are not viable. Potentially VGAT inhibitors
are a target for the treatment of epilepsy.

GABA Metabolism

GABA is metabolized intracellularly via the tricarboxylic acid cycle in mitochondria by GABA-aminotransferase (GABA-T) to
succinic semialdehyde then oxidized by succinic semialdehyde dehydrogenase to succinic acid.
The metabolic pathway 2-ketoglutarate—glutamate—GABA—succinate is known as the GABA shunt, a pathway which bypasses
part of the normal tricarboxylic cycle in brain. Up to 30% of the turnover of the tricarboxylic acid cycle in brain is via the GABA
shunt, illustrating the importance of GABA metabolism in the overall metabolism of the brain. Different pools of GABA with
different turnover rates and pool sizes are known to occur in the brain (Waagepetersen et al., 2003). Indeed, it is thought that only a
fraction of GABA in the brain is neurotransmitter GABA, with the remainder located in cell bodies as a metabolic pool. Inhibitors of
GABA metabolism can increase brain levels of GABA up to 10-fold and act as anticonvulsants. This provides evidence of the rapid
turnover of GABA in the brain.
The most well researched GABA-T inhibitor is g-vinyl-GABA (Fig. 8, Vigabatrin, Sabril) that is used as an anticonvulsant. It was
rationally designed as a suicide substrate binding irreversibly to the enzyme (Metcalf, 1979).

GABA Beyond the Central Nervous System

Although exogenous GABA is not generally considered to pass the blood–brain-barrier, orally administered GABA does have effects
that suggest it can alter brain function (Boonstra et al., 2015). GABA is an important constituent of most plants where it plays an
important role in plant metabolism and as a signaling molecule modulating anion flux (Rabbani et al., 2011). As such, GABA is
found in plant derived foodstuffs and herbal medicines. It occurs alongside numerous agents that can influence GABA function in
the brain, such as flavonoids (Hanrahan et al., 2015). A range of plant-derived anxiolytic medicines contains GABA modulating
agents (Savage et al., 2018).
GABA is widely available as a dietary supplement (Boonstra et al., 2015). Many people report that these supplements reduced
anxiety and improve sleep quality. Oral GABA has been shown to improve sleep (Yamatsu et al., 2016) and memory (Thanapree-
dawat et al., 2013). GABA enriched chocolate has a stress reducing effect (Nakamura et al., 2009). The levels of GABA in tea may be
considerably enhanced by anaerobic fermentation. Such GABA tea has been reported to reduce blood pressure, promote sleep, and
reduce depression (Teng et al., 2017; Zhao et al., 2015).
These effects of exogenous GABA may reflect actions on GABA receptors in the peripheral nervous system and interactions with
other agents that influence GABA receptors. Endogenous GABA is important in gastrointestinal function (Auteri et al., 2015) and the
effects of exogenous GABA may involve the enteric nervous system (Boonstra et al., 2015).
GABA occurs in high levels in insulin-producing b-cells of the pancreas. These cells release GABA to act on GABA receptors on
neighboring islet a-cells to inhibit the secretion of glucagon that counteracts the effects of insulin (Feng et al., 2017). These cells may
represent another site of action of exogenous GABA.
GABA is an important neurotransmitter in insects and insect GABA receptors are key targets for insecticides (Casida and Durkin,
2015). Whereas insect GABA receptors do differ from mammalian GABA receptors sufficiently to enable insecticides selectively
targeting insects to be produced, such as pyrethroids, toxicity to honey bees may be a problem (Oliver et al., 2015).

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GABA, The Major Inhibitory Neurotransmitter in the Brain 13

Conclusion

For such a simple molecule, GABA has many and varied complex functions. This research module concentrates on the role of GABA
as the major neurotransmitter in the brain. It emphasizes the range of agents that can influence the action of GABA as a
neurotransmitter and the effects that such agents can have on brain function. Agents that have increasingly selective actions on
GABA function are targets for the development of new drugs.

Acknowledgment

The authors are grateful to Dr. Ken Mewett for his helpful comments.

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